Berk2010-Group
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Amy N. Kristofferson 17:15, 7 June 2010 (EDT)
First step of Manual Assembly
Ligation:
Followed this protocol with the following amendments:
Mastermix
- 6.5uL ddH2O
- 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
- 0.5uL T4 DNA Ligase
DNA
- 3uL Lefty vector
- 3uL Right vector
Zymo:
Eluted with 10uL of H2O. Products are labeled 7+11 L, 9+8 L, 9+20 L.
Digestion:
Lefty MM added to 9+8, 7+11, 9+20
Right MM added to 7+23, 8+6, 7+5
They should be done incubating at 3:48pm.
Lefty MasterMix (for 4uL miniprep DNA)
- 4uL water
- 1uL of NEB2
- 0.5uL XhoI
- 0.5uL BamH1
Righty MasterMix (for 4uL miniprep DNA)
- 4uL water
- 1uL of NEB2
- 0.5uL XhoI
- 0.5uL BglII
Christoph Neyer 16:54, 7 June 2010 (EDT)
Some of Tim's spreadsheet data (which clones have been chosen for 2ab assembly):
Chosen clone (June2) Chosen clone (June3) 4 AC 9 B10sbb20 <FokI-! 1 1 4 AC 10 B10sbb43 {rbs2_lamB>} 2 1 4 AC 16 B10sbb15 phiC31 attB 2 1 seq 4 AC 17 B10sbb14 rbs_phiC31> 3 1 5 4 AC 18 B10sbb13 <phiC31> 3 1 7 AK 2 B10sbb09 Tn5 3'TR 4 1 seq 7 AK 5 B10sbb06 piggieBac3'TR 4 1 7 AK 9 B10sbb20 <FokI-! 5 1 7 AK 10 B10sbb43 {rbs2_lamB>} 5 7 AK 11 Bjh1906 b1006 6 1 seq 7 AK 13 B10sbb18 <zf-> 7 1 7 AK 14 B10sbb17 <zf+> 8 1 7 AK 19 B10sbb12 sleeping_beauty_3'TR 8 1 7 AK 23 B10sbb42 {rbs_pelB>} 9 d 10 7 AK 24 Bjh2294 Self_Lysis_Device 9 c 9 CA 3 Bjh1858 <NLS! 10 1 seq 9 CA 8 ig114 {AraC-Pbad} 11 1 9 CA 11 Bjh1906 b1006 11 a seq 9 CA 13 B10sbb18 <zf-> 11 1 9 CA 20 B10sbb44 Promoter_rbs_GenR 12 2 6 9 CA 23 B10sbb42 {rbs_pelB>} 13 a seq 5 CK 3 Bjh1858 <NLS! 13 1 seq 5 CK 8 ig114 {AraC-Pbad} 13 1 5 CK 15 B10sbb16 phiC31 attP 14 1 seq 5 CK 20 B10sbb44 Promoter_rbs_GenR 15 2 5 5 CK 23 B10sbb42 {rbs_pelB>} 16 1 seq 6 KA 2 B10sbb09 Tn5 3'TR 17 2 seq 6 KA 4 B10sbb07 <tn5> 17 2 6 KA 5 B10sbb06 piggieBac3'TR 18 2 6 KA 7 B10sbb04 <piggyBac! 18 2 6 KA 11 Bjh1906 b1006 19 1 seq 6 KA 12 B10sbb19 <FokI+! 19 2 seq 6 KA 13 B10sbb18 <zf-> 20 2 6 KA 19 B10sbb12 sleeping_beauty_3'TR 20 2 6 KA 22 B10sbb10 <SB100x! 21 1 6 KA 23 B10sbb42 {rbs_pelB>} 22 2 11 6 KA 24 Bjh2294 Self_Lysis_Device 23 a 8 KC 1 B10sbb08 Tn5 5'TR 23 1 seq 8 KC 4 B10sbb07 <tn5> 23 2 8 KC 6 B10sbb05 piggieBac_5'TR 23 1 8 KC 17 B10sbb14 rbs_phiC31> 24 2 8 KC 18 B10sbb13 <phiC31> 24 1 6 8 KC 21 B10sbb11 sleeping_beauty_5'TR 24 1
Tim Hsiau 19:48, 2 June 2010 (EDT)
Tim Hsiau 16:16, 1 June 2010 (EDT)
EcoXho's for the first round of assembly (ecobammed into assembly vectors)
appropriate fragment lengths
layout on ecoXho gel FRAGMENTS part size 1 6-2 1307 1605 35 2 6-11 1318 1605 46 3 6-2 1307 1605 35 4 6-11 1318 1605 46 5 6-4 2703 1605 1431 6 6-12 1869 1605 597 7 6-4 2703 1605 1431 8 6-12 1869 1605 597 9 6-5 1513 1605 241 10 6-13 1671 1605 399 11 6-5 1513 1605 241 12 6-13 1671 1605 399 13 6-7 3060 1605 1788 14 6-19 1500 1605 228 15 6-7 3060 1605 1788 16 6-19 1500 1605 228 17 6-22 2298 1605 1026 18 7-9 1797 1680 597 19 6-22 2298 1605 1026 20 7-10 1300 1680 100 21 6-23 1363 1605 91 22 7-11 1246 1680 46 23 6-23 1363 1605 91 24 7-13 1599 1680 399 25 6-24 #N/A 1605 #N/A 26 7-14 1599 1680 399 27 6-24 #N/A 1605 #N/A 28 7-19 1428 1680 228 29 7-2 1235 1680 35 30 7-23 1291 1680 91 31 7-5 1441 1680 241 32 7-24 #N/A 1680 #N/A 33 7-24 #N/A 1680 #N/A 34 5-23 1291 1473 91 35 5-3 1233 1473 33 36 5-23 1291 1473 91 37 5-3 1233 1473 33 38 8-1 1307 1473 35 39 5-8 2444 1473 1244 40 8-1 1307 1473 35 41 5-8 2444 1473 1244 42 8-4 2703 1473 1431 43 5-15 1290 1473 90 44 8-4 2703 1473 1431 45 5-20 2023 1473 823 46 8-6 1591 1473 319 47 5-20 2023 1473 823 48 8-6 1591 1473 319 1 8-17 3117 1473 1845 2 4-10 1166 1679 100 3 8-17 3117 1473 1845 4 4-10 1166 1679 100 5 8-18 3090 1473 1818 6 4-16 1156 1679 90 7 8-18 3090 1473 1818 8 4-16 1156 1679 90 9 8-21 1504 1473 232 10 4-17 2911 1679 1845 11 8-21 1504 1473 232 12 4-17 2911 1679 1845 13 4-9 1663 1679 597 14 4-18 2884 1679 1818 15 4-9 1663 1679 597 16 4-18 2884 1679 1818 17 9-3 1099 1605 33 18 9-20 1889 1605 823 19 9-3 1099 1605 33 20 9-20 1889 1605 823 21 9-8 2310 1605 1244 22 9-23 1157 1605 91 23 9-8 2310 1605 1244 24 9-23 1157 1605 91 25 9-11 1112 1605 46 26 27 9-11 1112 1605 46 28 29 9-13 1465 1605 399 30 31 9-13 1465 1605 399 32 33 34 35